Molecular mechanisms of enzymatic post-translational modifications at the membrane

Thesis

Post-translational modifications (PTMs) of proteins modulate their subcellular localisation, protein-protein interactions, and stability. Reversible PTMs, which include ubiquitination and S-palmitoylation, dynamically regulate cellular homeostasis and signalling processes in response to environmental stimuli. Cells must precisely regulate PTMs to prevent the development and progression of diseases, including cancer, neurodegenerative disorders, and immune dysfunction. This thesis uses cryo-electron microscopy (cryo-EM) to elucidate the molecular mechanism of ubiquitination, catalysed by a novel membrane-bound ubiquitin ligase termed the MMM complex, and S-palmitoylation, catalysed by DHHC20.

The key Hedgehog signal transducer and oncoprotein Smoothened is targeted for degradation by the covalent attachment of a small protein, ubiquitin (termed ubiquitination), catalysed by the MMM complex. This thesis presents high-resolution cryo-EM structures of the MMM complex, a membrane-spanning E3 ligase complex composed of MEGF8, MOSMO and MGRN1, which represents the first structure of a larger group of transmembrane ubiquitin ligases that regulate diverse receptors, such as Frizzled proteins in WNT signalling. These studies shed light on how membrane-based ubiquitin ligase assemblies target folded receptors for ubiquitination, thus fine-tuning signal transduction.

A palmitoyl moiety is covalently attached to a cysteine thiol in S-palmitoylation, catalysed by the DHHC family of integral membrane acyltransferases. Although DHHC20 is increasingly recognised as a potential therapeutic target, the molecular mechanism underlying DHHC-mediated palmitoylation remains incompletely understood, posing a barrier to the rational development of DHHC-targeted therapies. Thus, in this thesis, multiple high-resolution cryo-EM structures of DHHC20 in different functional states were determined, enabling us to visualise catalytic intermediates of the palmitoylation cycle which were previously unresolved.

Authors: Williams, C

• DOI: 10.5287/ora-j0m4reprk
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Hedgehog signalling; post-translational modifications; ubiquitination; palmitoylation; E3 ligase; DHHC20; cryo-electron microscopy; membrane protein; acylation
• Subjects: structural biology